Profile of Embedded Validity Indicators in Criminal Defendants with Verified Valid Neuropsychological Test Performance.

OBJECTIVE: Few studies have examined the use of embedded validity indicators (EVIs) in criminal-forensic practice settings, where judgements regarding performance validity can carry severe consequences for the individual and society. This study sought to examine how various EVIs perform in criminal defendant populations, and determine relationships between EVI scores and intrapersonal variables thought to influence performance validity. METHOD: Performance on 16 empirically established EVI cutoffs were examined in a sample of 164 criminal defendants with valid performance who were referred for forensic neuropsychological evaluation. Subsequent analyses examined the relationship between EVI scores and intrapersonal variables in 83 of these defendants. RESULTS: Half of the EVIs (within the Wechsler Adult Intelligence Scale Digit Span Total, Conners' Continuous Performance Test Commissions, Wechsler Memory Scale Logical Memory I and II, Controlled Oral Word Association Test, Trail Making Test Part B, and Stroop Word and Color) performed as intended in this sample. The EVIs that did not perform as intended were significantly influenced by relevant intrapersonal variables, including below-average intellectual functioning and history of moderate-severe traumatic brain injury and neurodevelopmental disorder. CONCLUSIONS: This study identifies multiple EVIs appropriate for use in criminal-forensic settings. However, based on these findings, practitioners may wish to be selective in choosing and interpreting EVIs for forensic evaluations of criminal court defendants.

Effect of a Clinical Case-Study Course on Physician Assistant and Pharmacy Students' Interprofessional Care Competencies.

PURPOSE: To examine how the implementation of a year-long interprofessional clinical case course for pharmacy and physician assistant (PA) students affects student self-reported interprofessional collaboration-related competencies in 6 skill areas (communication, collaboration, roles and responsibilities, collaborative patient/family-centered approach, conflict management/resolution, and team functioning) and whether outcomes differed between the 2 professions. METHODS: Pharmacy and PA students completed the Interprofessional Collaborative Competency Attainment Survey (ICCAS) at the beginning and end of a year-long interprofessional, team-based clinical case course. Survey results were compared using a mixed-design analysis of variance model to determine the effect the course had on students' self-reported competencies of interprofessional care and whether the outcomes differed between student groups. RESULTS: One-hundred fifteen students completed both the presurvey and postsurvey. Significant improvement in student self-reported team-based behaviors were noted in 11 of the 20 ICCAS items, and results were similar among student groups. CONCLUSION: This study demonstrates that an interactive, interprofessional clinical case course can positively change student self-reported team-based behaviors.

Psychosocial factors associated with treatment outcomes in women with obesity and major depressive disorder who received behavioral activation for depression.

Behavioral activation is an empirically supported treatment for depression, but much is unknown about factors associated with treatment response. The present study aimed to determine whether baseline levels and subsequent changes in psychosocial factors were associated with improvement in depression in women with comorbid obesity who received behavioral activation treatment for depression and a lifestyle intervention. Multilevel modeling was used to estimate the associations between psychosocial factors and change in depression scores during the first 10 weeks of treatment and associations between changes in psychosocial factors from baseline to 6-month follow-up and change in depression over the same time period. No baseline psychosocial factors were associated with depression improvement during treatment (p = 0.110-0.613). However, greater improvement in hedonic capacity (p = 0.001), environmental reward (p = 0.004), and social impairment (p = 0.012) were associated with greater reductions in depression over 6 months. Findings highlight the differential relationship specific psychosocial factors have with depression treatment outcomes.

Correlates of Active Videogame Use in Children.

OBJECTIVES: Active videogames (AVGs) could provide a novel approach to increasing physical activity and decreasing sedentary activity in children, but little is known about which children are likely to use AVGs. This study examined whether youth demographics, social support, and AVG engagement influence use of AVGs and physical activity. MATERIALS AND METHODS: A diverse sample of youth participants (42.4% non-Hispanic white), aged 8-14 years (n = 85), who owned an AVG console, completed surveys, wore an activity monitor, and logged AVG use for 1 week. Regression analyses were used to examine variables associated with daily AVG minutes and to examine the relationship between daily AVG minutes and daily steps. RESULTS: Older and non-Hispanic white children played AVGs for fewer minutes per day (P's < 0.03). Greater peer support for playing AVGs was associated with greater daily AVG minutes (P = 0.003). Daily AVG minutes were not associated with daily steps. CONCLUSIONS: Results suggest that younger children and children who do not identify as non-Hispanic white may be more open to playing AVGs. Targeting social support in AVG interventions may increase time spent playing AVGs.

Feasibility of Pairing Behavioral Activation With Exercise for Women With Type 2 Diabetes and Depression: The Get It Study Pilot Randomized Controlled Trial.

Major depressive disorder is often comorbid with diabetes and associated with worse glycemic control. Exercise improves glycemic control and depression, and thus could be a parsimonious intervention for patients with comorbid diabetes and major depression. Because patients with diabetes and comorbid depression are often sedentary and lack motivation to exercise, we developed a group exercise intervention that integrates strategies from behavioral activation therapy for depression to increase motivation for and enjoyment of exercise. We conducted a 6-month pilot randomized controlled trial to test the feasibility of the behavioral activation exercise intervention (EX) for women with diabetes and depression. Of the 715 individuals who contacted us about the study, 29 participants were randomized to the EX condition or an enhanced usual care condition (EUC), which represents 4.1% of participants who initially contacted us. Inclusion criteria made recruitment challenging and limits the feasibility of recruiting women with diabetes and depression for a larger trial of the intervention. Retention was 96.5% and 86.2% at 3 and 6months. Participants reported high treatment acceptability; use of behavioral activation strategies and exercise class attendance was acceptable. No condition differences were observed for glycemic control, depressive symptoms, and physical activity, though depressive symptoms and self-reported physical activity improved over time. Compared to participants in the EUC condition, participants in the EX condition reported greater exercise enjoyment and no increase in avoidance behavior over time. Using behavioral activation strategies to increase exercise is feasible in a group exercise setting. However, whether these strategies can be delivered in a less intensive manner to a broader population of sedentary adults, for greater initiation and maintenance of physical activity, deserves further study.

An online social network to increase walking in dog owners: a randomized trial.

PURPOSE: Encouraging dog walking may increase physical activity in dog owners. This cluster-randomized controlled trial investigated whether a social networking Web site (Meetup™) could be used to deliver a multicomponent dog walking intervention to increase physical activity. METHODS: Sedentary dog owners (n = 102) participated. Eight neighborhoods were randomly assigned to the Meetup™ condition (Meetup™) or a condition where participants received monthly e-mails with content from the American Heart Association regarding increasing physical activity. The Meetup™ intervention was delivered over 6 months and consisted of newsletters, dog walks, community events, and an activity monitor. The primary outcome was steps; secondary outcomes included social support for walking, sense of community, perceived dog walking outcomes, barriers to dog walking, and feasibility of the intervention. RESULTS: Mixed-model analyses examined change from baseline to postintervention (6 months) and whether change in outcomes differed by condition. Daily steps increased over time (P = 0.04, d = 0.28), with no differences by condition. The time-condition interaction was significant for the perceived outcomes of dog walking (P = 0.04, d = 0.40), such that the Meetup™ condition reported an increase in the perceived positive outcomes of dog walking, whereas the American Heart Association condition did not. Social support, sense of community, and dog walking barriers did not significantly change. Meetup™ logins averaged 58.38 per week (SD, 11.62). Within 2 months of the intervention ending, organization of the Meetup™ groups transitioned from the study staff to Meetup™ members. CONCLUSIONS: Results suggest that a Meetup™ group is feasible for increasing physical activity in dog owners. Further research is needed to understand how to increase participation in the Meetup™ group and facilitate greater connection among dog owners.

Critical role of zinc ion on E. coli glutamyl-queuosine-tRNA(Asp) synthetase (Glu-Q-RS) structure and function.

Glutamyl-queuosine-tRNA(Asp) synthetase (Glu-Q-RS) and glutamyl-tRNA synthetase (GluRS), differ widely by their function although they share close structural resemblance within their catalytic core of GluRS. In particular both Escherichia coli GluRS and Glu-Q-RS contain a single zinc-binding site in their putative tRNA acceptor stem-binding domain. It has been shown that the zinc is crucial for correct positioning of the tRNA(Glu) acceptor-end in the active site of E. coli GluRS. To address the role of zinc ion in Glu-Q-RS, the C101S/C103S Glu-Q-RS variant is constructed. Energy dispersive X-ray fluorescence show that the zinc ion still remained coordinated but the variant became structurally labile and acquired aggregation capacity. The extent of aggregation of the protein is significantly decreased in presence of the small substrates and more particularly by adenosine triphosphate. Addition of zinc increased significantly the solubility of the variant. The aminoacylation assay reveals a decrease in activity of the variant even after addition of zinc as compared to the wild-type, although the secondary structure of the protein is not altered as shown by the Fourier transform infrared spectroscopy study.

Fusion with anticodon binding domain of GluRS is not sufficient to alter the substrate specificity of a chimeric Glu-Q-RS.

Glutamyl-queuosine-tRNA(Asp) synthetase (Glu-Q-RS) is a paralog of glutamyl-tRNA synthetase (GluRS) and is found in more than forty species of proteobacteria, cyanobacteria, and actinobacteria. Glu-Q-RS shows striking structural similarity with N-terminal catalytic domain of GluRS (NGluRS) but it lacks the C-terminal anticodon binding domain (CGluRS). In spite of structural similarities, Glu-Q-RS and NGluRS differ in their functional properties. Glu-Q-RS glutamylates the Q34 nucleotide of the anticodon of tRNA(Asp) whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNA(Glu). Since NGluRS is able to catalyze aminoacylation of only tRNA(Glu) the glutamylation capacity of tRNA(Asp) by Glu-Q-RS is surprising. To understand the substrate specificity of Glu-Q-RS we undertook a systemic approach by investigating the biophysical and biochemical properties of the NGluRS (1-301), CGluRS (314-471) and Glu-Q-RS-CGluRS, (1-298 of Glu-Q-RS fused to 314-471 from GluRS). Circular dichroism, fluorescence spectroscopy and differential scanning calorimetry analyses revealed absence of N-terminal domain (1-298 of Glu-Q-RS) and C-terminal domain (314-471 from GluRS) communication in chimera, in contrast to the native full length GluRS. The chimeric Glu-Q-RS is still able to aminoacylate tRNA(Asp) but has also the capacity to bind tRNA(Glu). However the chimeric protein is unable to aminoacylate tRNA(Glu) probably as a consequence of the lack of domain-domain communication.

A tRNA-independent mechanism for transamidosome assembly promotes aminoacyl-tRNA transamidation.

Many bacteria lack genes encoding asparaginyl- and/or glutaminyl-tRNA synthetase and consequently rely on an indirect path for the synthesis of both Asn-tRNA(Asn) and Gln-tRNA(Gln). In some bacteria such as Thermus thermophilus, efficient delivery of misacylated tRNA to the downstream amidotransferase (AdT) is ensured by formation of a stable, tRNA-dependent macromolecular complex called the Asn-transamidosome. This complex enables direct delivery of Asp-tRNA(Asn) from the non-discriminating aspartyl-tRNA synthetase to AdT, where it is converted into Asn-tRNA(Asn). Previous characterization of the analogous Helicobacter pylori Asn-transamidosome revealed that it is dynamic and cannot be stably isolated, suggesting the possibility of an alternative mechanism to facilitate assembly of a stable complex. We have identified a novel protein partner called Hp0100 as a component of a stable, tRNA-independent H. pylori Asn-transamidosome; this complex contains a non-discriminating aspartyl-tRNA synthetase, AdT, and Hp0100 but does not require tRNA(Asn) for assembly. Hp0100 also enhances the capacity of AdT to convert Asp-tRNA(Asn) into Asn-tRNA(Asn) by ∼35-fold. Our results demonstrate that bacteria have adopted multiple divergent methods for transamidosome assembly and function.

Protein analysis by dynamic light scattering: methods and techniques for students.

Dynamic light scattering (DLS) analyses are routinely used in biology laboratories to detect aggregates in macromolecular solutions, to determine the size of proteins, nucleic acids, and complexes or to monitor the binding of ligands. This article is written for graduate and undergraduate students with access to DLS and for faculty members who wish to incorporate DLS into a lab activity, a practical course or research. It reviews the basic concepts of light scattering measurements and addresses four critical aspects of the analysis and interpretation of DLS results. To ensure reproducible quantitative data, attention should be paid to controlling the preparation and handling of proteins or assemblies because variations in the state of aggregation, induced by minor changes in experimental condition or technique, might compromise DLS results and affect protein activity. Variables like temperature, solvent viscosity, and inter-particle interactions may also influence particle size determination. Every point is illustrated by case studies, including a commercially available albumin, a small RNA virus isolated from plants, as well as four soluble proteins and a ribonucleoprotein assembly purified and characterized by students in the frame of their master degree.

The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code.

Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.

Crystal structure of the archaeal asparagine synthetase: interrelation with aspartyl-tRNA and asparaginyl-tRNA synthetases.

Asparagine synthetase A (AsnA) catalyzes asparagine synthesis using aspartate, ATP, and ammonia as substrates. Asparagine is formed in two steps: the ß-carboxylate group of aspartate is first activated by ATP to form an aminoacyl-AMP before its amidation by a nucleophilic attack with an ammonium ion. Interestingly, this mechanism of amino acid activation resembles that used by aminoacyl-tRNA synthetases, which first activate the α-carboxylate group of the amino acid to form also an aminoacyl-AMP before they transfer the activated amino acid onto the cognate tRNA. In a previous investigation, we have shown that the open reading frame of Pyrococcus abyssi annotated as asparaginyl-tRNA synthetase (AsnRS) 2 is, in fact, an archaeal asparagine synthetase A (AS-AR) that evolved from an ancestral aspartyl-tRNA synthetase (AspRS). We present here the crystal structure of this AS-AR. The fold of this protein is similar to that of bacterial AsnA and resembles the catalytic cores of AspRS and AsnRS. The high-resolution structures of AS-AR associated with its substrates and end-products help to understand the reaction mechanism of asparagine formation and release. A comparison of the catalytic core of AS-AR with those of archaeal AspRS and AsnRS and with that of bacterial AsnA reveals a strong conservation. This study uncovers how the active site of the ancestral AspRS rearranged throughout evolution to transform an enzyme activating the α-carboxylate group into an enzyme that is able to activate the ß-carboxylate group of aspartate, which can react with ammonia instead of tRNA.

Gln-tRNAGln synthesis in a dynamic transamidosome from Helicobacter pylori, where GluRS2 hydrolyzes excess Glu-tRNAGln.

In many bacteria and archaea, an ancestral pathway is used where asparagine and glutamine are formed from their acidic precursors while covalently linked to tRNA(Asn) and tRNA(Gln), respectively. Stable complexes formed by the enzymes of these indirect tRNA aminoacylation pathways are found in several thermophilic organisms, and are called transamidosomes. We describe here a transamidosome forming Gln-tRNA(Gln) in Helicobacter pylori, an ε-proteobacterium pathogenic for humans; this transamidosome displays novel properties that may be characteristic of mesophilic organisms. This ternary complex containing the non-canonical GluRS2 specific for Glu-tRNA(Gln) formation, the tRNA-dependent amidotransferase GatCAB and tRNA(Gln) was characterized by dynamic light scattering. Moreover, we observed by interferometry a weak interaction between GluRS2 and GatCAB (K(D) = 40 ± 5 µM). The kinetics of Glu-tRNA(Gln) and Gln-tRNA(Gln) formation indicate that conformational shifts inside the transamidosome allow the tRNA(Gln) acceptor stem to interact alternately with GluRS2 and GatCAB despite their common identity elements. The integrity of this dynamic transamidosome depends on a critical concentration of tRNA(Gln), above which it dissociates into separate GatCAB/tRNA(Gln) and GluRS2/tRNA(Gln) complexes. Ester bond protection assays show that both enzymes display a good affinity for tRNA(Gln) regardless of its aminoacylation state, and support a mechanism where GluRS2 can hydrolyze excess Glu-tRNA(Gln), ensuring faithful decoding of Gln codons.

Crystal structure of a transfer-ribonucleoprotein particle that promotes asparagine formation.

Four out of the 22 aminoacyl-tRNAs (aa-tRNAs) are systematically or alternatively synthesized by an indirect, two-step route requiring an initial mischarging of the tRNA followed by tRNA-dependent conversion of the non-cognate amino acid. During tRNA-dependent asparagine formation, tRNA(Asn) promotes assembly of a ribonucleoprotein particle called transamidosome that allows channelling of the aa-tRNA from non-discriminating aspartyl-tRNA synthetase active site to the GatCAB amidotransferase site. The crystal structure of the Thermus thermophilus transamidosome determined at 3 A resolution reveals a particle formed by two GatCABs, two dimeric ND-AspRSs and four tRNAs(Asn) molecules. In the complex, only two tRNAs are bound in a functional state, whereas the two other ones act as an RNA scaffold enabling release of the asparaginyl-tRNA(Asn) without dissociation of the complex. We propose that the crystal structure represents a transient state of the transamidation reaction. The transamidosome constitutes a transfer-ribonucleoprotein particle in which tRNAs serve the function of both substrate and structural foundation for a large molecular machine.

Arc1p: anchoring, routing, coordinating.

Accurate synthesis of aminoacyl-tRNAs (aa-tRNA) by aminoacyl-tRNA synthetases (aaRS) is an absolute requirement for errorless decoding of the genetic code and is studied since more than four decades. In all three kingdoms of life aaRSs are capable of assembling into multi-enzymatic complexes that are held together by auxiliary non-enzymatic factors, but the role of such macromolecular assemblies is still poorly understood. In the yeast Saccharomyces cerevisiae, Arc1p holds cytosolic methionyl-tRNA synthetase ((c)MRS) and glutamyl-tRNA synthetase ((c)ERS) together and plays an important role in fine tuning several cellular processes like aminoacylation, translation and carbon source adaptation.

Yeast mitochondrial Gln-tRNA(Gln) is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS.

It is impossible to predict which pathway, direct glutaminylation of tRNA(Gln) or tRNA-dependent transamidation of glutamyl-tRNA(Gln), generates mitochondrial glutaminyl-tRNA(Gln) for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNA(Gln) has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNA(Gln) is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA-dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase ((c)ERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the mitochondrial mischarged glutamyl-tRNA(Gln) substrate for the AdT. We show that dual localization of (c)ERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for (c)ERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of (c)ERS to satisfy a higher demand of mitochondrial glutaminyl-tRNA(Gln) for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments.

Isolation, crystallization and preliminary X-ray analysis of the transamidosome, a ribonucleoprotein involved in asparagine formation.

Thermus thermophilus deprived of asparagine synthetase synthesizes Asn on tRNA(Asn) via a tRNA-dependent pathway involving a nondiscriminating aspartyl-tRNA synthetase that charges Asp onto tRNA(Asn) prior to conversion of the Asp to Asn by GatCAB, a tRNA-dependent amidotransferase. This pathway also constitutes the route of Asn-tRNA(Asn) formation by bacteria and archaea deprived of asparaginyl-tRNA synthetase. The partners involved in tRNA-dependent Asn formation in T. thermophilus assemble into a ternary complex called the transamidosome. This particule produces Asn-tRNA(Asn) in the presence of free Asp, ATP and an amido-group donor. Crystals of the transamidosome from T. thermophilus were obtained in the presence of PEG 4000 in MES-NaOH buffer pH 6.5. They belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 115.9, b = 214.0, c = 127.8 A, beta = 93.3 degrees . A complete data set was collected to 3 A resolution. Here, the isolation and crystallization of the transamidosome from T. thermophilus and preliminary crystallographic data are reported.

Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed with L-glutamate: structural elements mediating tRNA-independent activation of glutamate and glutamylation of tRNAAsp anticodon.

Glutamyl-queuosine tRNA(Asp) synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA(Asp). Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA(Glu), Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3' accepting end of the cognate tRNA(Glu), Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA(Asp). In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS.Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA(Asp) among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA(Asp) that determine recognition by Glu-Q-RS. The analyses made on tRNA(Asp) and tRNA(Asn) show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.

Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants.

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNA(Gln) is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNA(Gln) is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNA(Gln), which is the only tRNA(Gln) present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNA(Gln) formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts.

tRNA-dependent asparagine formation in prokaryotes: characterization, isolation and structural and functional analysis of a ribonucleoprotein particle generating Asn-tRNA(Asn).

In some living organisms the 20 aa-tRNA species participating in protein synthesis are not charged by a complete set of 20 aminoacyl-tRNA synthetases. In prokaryotes, the deficiency of asparaginyl- and/or glutaminyl-tRNA synthetases is compensated by another aminoacyl-tRNA synthetase of relaxed specificity that mischarges the orphan tRNA and by an enzyme that converts the amino acid into that homologous to the tRNA. In Thermus thermophilus Asn-tRNA(Asn) is formed indirectly via a two-step pathway whereby tRNA(Asn) is mischarged with Asp that will subsequently be amidated into Asn by an amidotransferase. The non-discriminating aspartyl-tRNA synthetase, the trimeric GatCAB tRNA-dependent amidotransferase and the tRNA(Asn) promoting this pathway assemble into a ribonucleoprotein particle termed transamidosome. This article deals with the methods and techniques employed to clone the genes encoding the enzymes and the tRNA involved in this pathway, to express them in Escherichia coli, to isolate them on a large scale, and to transcribe and produce mg quantities of pure tRNA(Asn)in vitro. The approaches designed especially for this system include (i) clustering of the ORFs encoding the subunits of the heterotrimeric GatCAB that are sprinkled in the genome into an artificial operon, and (ii) the self-cleavage of the tRNA(Asn) transcript starting with U in 5' position through fusion with a hammerhead ribozyme. Further, the crystallization of the free enzymes is described and the characterization of their assembly with tRNA(Asn) into a ribonucleoprotein particle, as well as the investigation of the catalytic mechanism of Asn-tRNA(Asn) formation by the complex are reported.